Mismatch repair plays a key role in genetic stabilization, with inactivation of the human pathway being the cause of several forms of cancer. This system has been conserved during evolution, and the E. coil methyl-directed reaction has served as the paradigm for study of the reaction in higher cells. Although bacterial mismatch repair has been reconstituted in a pure system, numerous questions remain concerning the mechanism of the reaction. This proposal addresses several of these issues. Mismatch recognition by MutS is responsible for initiation of repair, but function of the MutS ATPase is poorly understood. Using substrate binding and pre-steady-state kinetic methods, we will further clarify the nature of the interactions between the MutS nucleotide and DNA binding centers. A number of multiprotein.DNA assemblies have been implicated in mismatch repair, but the molecular nature of the MutL.MutS, MutH.MutL.MutS, and DNA helicase II.MutL.MutS complexes remains undefined. A second goal of this project is to establish the nature of these multi-protein DNA assemblies. This phase of the work will also address the significance of the specific interaction between the beta replication clamp and MutS. Since this interaction may be indicative of a special affiliation of the mismatch repair system with the replication apparatus, we will test the clamp loader and the beta clamp for potential modulatory effects on the initiation and excision steps of mismatch repair, and assess potential regulatory effects of MutS, MutL, and other repair proteins on the elongation activity of DNA polymerase III holoenzyme in response to presence of a mismatch within the primer-template. Basic features of the excision step of mismatch repair have been established, but the mechanism that underlies this bidirectional reaction is not understood. The third aim of this project is to further clarify the molecular details of this complex reaction with respect to intermediates, recycling of repair activities, and the basis of excision termination upon mismatch removal.